The crucial role of the p53 protein in tumor suppression is underscored by the findings that p53 is mutated in over half of all human cancers and that p53-deficient mice universally develop cancer. While p53 suppresses cancer by inducing cell cycle arrest, senescence or apoptosis in response to stress signals, its molecular mechanism of action in vivo remains unclear. p53 displays activity as a transcriptional activator that can induce numerous target genes, but it also has a variety of other biochemical activities, in regulating transcriptional repression, DNA recombination, and mitochondrial membrane integrity. To clarify the p53 molecular activities most relevant for tumor suppression, we previously generated knock-in mice expressing a p53 mutant, p5325,26, that is severely impaired for the transactivation of most genes but retains the ability to robustly transactivate a small set of targets, such as Bax. Moreover, p5325,26 retains biological activity in senescence and tumor suppression. These observations suggest that p5325,26 tumor suppressor function is accounted for by 1) residual strong transactivation of target genes like Bax, 2) very low level expression of most other p53 targets, or 3) another activity of p53. Here, these possibilities will be distinguished by generating and analyzing novel p53 knock-in mutant strains. By examining knock-in mice expressing a mutant totally defective for transactivation through mutation of both p53 transactivation domains, p5325,26,53,54, the importance of transactivation for p53 effector functions of cell cycle arrest, senescence and apoptosis, as well as for tumor suppression in tissues in vivo, will be assessed. Additionally, the roles of transactivation for p53- induced tumor regression in response to cancer therapy will be determined. These studies using knock- in mice, in which mutants are expressed under the control of the native p53 promoter, and in which normal cells can be studied both ex vivo and in the physiological context of the organism, should provide definitive insight into the mechanism of p53 action in tumor suppression and therapeutic responses. Understanding whether activation of the full complement of p53 target genes or a subset of these genes is important for tumor suppression, as well as whether other p53 biochemical activities are important for tumor suppression, will lead to the development of better cancer therapies. PUBLIC HEALTH RELEVANCE: In the US, approximately 1 in every 2-3 individuals will develop cancer, a disease of uncontrolled cellular proliferation that can be fatal. It is therefore very important to understand the basis of this disease in order to be able to more effectively treat and cure it. This research aims to elucidate how p53, a gene important for preventing cancer, acts, with the ultimate goal of devising better prevention and treatment strategies for cancer.